Alphaviruses are arthropod-vectored viruses that can cause human diseases ranging from mild febrile illness to severe arthralgia or fatal encephalitis. Due to several factors including the ease of their isolation, long-term stability and potential for high morbidity/mortality when administered by aerosol, several have been designated Category B Select Agents with potential for biowarfare/bioterrorism use. Unfortunately, no licensed vaccines or antiviral medications are available to combat these infections. Interruption of the initial interactions of viruses with cells is one of the most effective means of preventing infection and disease and an understanding of virus-infection receptor interactions is vital to design of infection-blocking drugs. However, little is known about the receptor interactions of alphaviruses that have not been adapted extensively to growth in cell culture. Using a novel vector system, we propose to examine simultaneously, the receptor interactions of multiple non-cell adapted alphaviruses representative of both New World and Old World strains. In the first Aim, we will investigate the utilization of previously characterized cell surface receptor molecules heparan sulfate and the high affinity laminin receptor using well-characterized cell lines. In the second Aim we will utilize our novel vector system and a recently-identified alphavirus receptor-negative cell line in a cDNA library screen to identify new molecules that can mediate alphavirus infection of cells. The results of these experiments will provide a comprehensive understanding of the receptor interactions of New World and Old World alphaviruses and provide a framework for design of infection-blocking antiviral drugs. PUBLIC HEALTH RELEVANCE: Our plan is to use a novel chimeric alphavirus replicon system in which a propagation-defective Venezuelan equine encephalitis virus genome is packaged in the structural proteins of different Old World and New World alphaviruses to compare the receptor utilization characteristics of low passage strains of these viruses. These comparisons will include direct evaluation of the capacity of previously-identified receptors (heparan sulfate, the high affinity laminin receptor) to mediate infection as well characterization of new receptors through use of a retrovirus expression library and alphavirus receptor-negative cells.